1. A GH antagonist fusion with GH binding protein is biologically active, shows delayed clearance and inhibits growth in rabbits
Ian Wilkinson, Sarbhendra Pradhananga, Jon Sayers, Pete Artymiuk, Michael Phipps & Richard Ross
Sheffield University, UK 1

2. Action of GH and Pegvisomant
B2036
PEG
GHBP 1 2
JAK2 1*
GHBP
GHBP X X
Ross et al. J Clin Endocrinol Metab ,86(4):

3. Growth Hormone Antagonist - The Unmet Need
Acromegaly: Current Treatment
Only 50% cured by surgery
Only 70% controlled by SMS analogue therapy
Pegvisomant
Controls >95% and well tolerated
Majority of patients require daily dosing
Requires high dose of 20-60mg per day
High cost of goods & very expensive: £55k / year
Challenges
Generate a potent long-acting antagonist
Weekly injections
Define an animal model to test human GH antagonist

4. Background: LR-Fusion Technology
Linking GH to GHBP (extracellular domain GHR) creates a long acting agonist
[Ref: Wilkinson et al., Nature Medicine 2007]
Our AFTTM fusion protein 1 2 3
Extracellular receptor
Intermolecular interaction
Flexible GHR
N-terminus
Ligand
ProFuseTM constituents
(expressed as single construct)
Physiological pool of protected ligand
(dynamic equilibrium)
Ligand available to bind to natively expressed receptor
(long acting in vivo effect)

5. LR-Fusion GH antagonist: Expressed & Purified
Two LR-fusions made with and without Gly-Ser linkers
Site two mutant with G120R
Site two and site one
SDS-PAGE gel of purified LR-fusions
kDa
100 75 50 37 25 20
Key:
M: Markers
1: Site 2 (+ linker)
2: Site 2 (no linker)
3: Site 1 & 2 (+ linker)
4: Site 1 & 2 (no linker

6. Site 2 mutant is an antagonist in dual Luciferase bioassay

7. Site 2 mutant antagonist shows delayed clearance in rat
Given at 1nmol/kg subcutaneously (sc) to 6 rats the LR fusion antagonist (site 2 only) showed delayed clearance with plasma half-life of 21.0hrs compared to GH at 1.2hrs
LR fusion antagonist can still be detected 10 days post injection
sc injection
iv injection

8. Site 2 mutant antagonist more potent than PEGvisomant in vivo
A single sc dose of GH antagonist resulted in decreased weight gain in growing NZ white rabbits over 12 days compared to vehicle control
Decrease in weight gain was greater than Pegvisomant given as 5 separate injections (Days 1, 2, 6, 7 & 8)
Pegvisomant 5 x sc dosing regime

9. Summary The LR fusion antagonist Proof of concept :
is an antagonist in vitro and in vivo
Is more potent than Pegvisomant in vivo
Proof of concept :
Delayed clearance demonstrated in rat
Block GH activity in bioassay
Growth inhibition demonstrated in rabbit growth model
Potential for weekly dosing
Preclinical work required:
Candidate optimisation in progress
Further develop rabbit model for proof of concept using IGF-I

10. Acknowledgements Berlin Group
Christian Strasburger
Zida Wu
Work colleagues Richard Ross Sarbendra Pradhananga Pete Artymiuk Jon Sayers Sue Justice Michael Phipps Munich Group Max Bielohuby Martin Bidlingmaier